7.5.4 Isolation and Identification
In view of the metabolic diversity within the species selective media are of
limited use in the isolation ofC. botulinumand identification is based on
the ability of typical colonies to produce toxin in culture.
C. botulinumwill often constitute only a small proportion of the total
microflora so enrichment or pre-incubation is necessary to improve the
chances of isolation. Sometimes enrichment cultures are heated prior to
incubation to eliminate non-sporeforming anaerobes. However, depend-
ing on the heating regime used, 80 1 C for 10 min is commonly cited, this
may also eliminate the less heat resistant strains ofC. botulinumand is
therefore often omitted.
After enrichment in a medium such as cooked meat broth at 30 1 C for
7 days, the culture is streaked on to fresh horse-blood or egg yolk agar
and incubated anaerobically for 3 days. Characteristic smooth colonies,
2–3 mm in diameter with an irregular edge and showing lipolytic activity
on egg-yolk agar (type G excepted) are transferred into a broth medium
to check for toxin production.
A technique has been described that simplifies this procedure by
incorporating antitoxin into the agar medium so that toxin-producing
colonies are surrounded by a zone of toxin–antitoxin precipitate.
Despite the development of a range ofin vitroimmunoassay proce-
dures for toxin, the mouse neutralization test (Figure 7.4), remains the
most sensitive (a typical lethal dose of toxin for a mouse is a few
picograms). However, the distressing nature of the test guarantees its
eventual replacement as soon as immunoassay amplification systems
have been sufficiently improved.
A suspect toxin extract is divided into three portions: one, to serve as
control, is heated at 100 1 C for 10 min to destroy any toxin present; a
second is treated with trypsin to activate any protoxin that may be there;
and a third is untreated. Each of the portions is injected intraperitoneally
into 2 mice and the mice observed over 4 days for the development of
typical symptoms of laboured breathing and the characteristic ‘wasp-
waist’ appearance. The presence of toxin is confirmed by protection of
mice with polyvalent antitoxin and the toxin type can be identified using
monovalent antisera.
7.5.5 Association with Foods
Four common features are discernible in outbreaks of botulism.
(1) The food has been contaminated at source or during processing
with spores or vegetative cells ofC. botulinum.Chapter 7 205